Without limiting the scope of the invention, its background is described in connection with intein mediated purification of protein.
U.S. Patent Application 2006/0141570 (filed Nov. 16, 2005) discloses purification of recombinant proteins performed by expressing in a host cell a fusion protein comprising a product protein domain, an intein, and at least one aggregator protein domain, wherein the aggregator protein domain comprises a protein that is capable of specific association with granules of polyhydroxyalkanoate (PHA).
European patent application EP 1117693 B1 (filed Sep. 30, 1999) discloses an in vitro method for producing a semi-synthetic fusion protein, whereby a target protein fused to an intein is selectively cleaved in a first step with a thiol reagent, forming a carboxyl-terminal thioester of the target protein and releasing the target protein from the intein. In a subsequent step, a desired, synthetic, protein or peptide having an amino-terminal cysteine is ligated to the target protein. Standard thiol-reagents such as DTT, or thiol-reagents optimized for ligation such as the odorless MESNA, may be used in the first step. The method is said to permit direct ligation of a desired peptide to a thioester bond that had linked a target protein to an intein. An in vivo variation of the method is said to permit production of a cytotoxic protein: a truncated, inactive, form of the protein fused to an intein is introduced in vivo, this fusion product is then selectively cleaved, and a synthetic protein or peptide is subsequently ligated at a carboxyl-terminal thioester of the target protein in order to restore the native activity of the cytotoxic protein.
European patent application EP 1151117 A4 (filed Aug. 10, 2005) discloses a method for the ligation of expressed proteins, which utilizes inteins, for example the RIR1 intein from Methanobacterium thermotrophicum. Constructs of the Mth RIR1 intein in which either the C-terminal asparagine or N-terminal cysteine of the intein are replaced with alanine enable the facile isolation of a protein with a specified N-terminal, for example, cysteine for use in the fusion of two or more expressed proteins. The method involves the steps of generating a C-terminal thioester-tagged target protein and a second target protein having a specified N-terminal via inteins, such as the modified Mth RIR1 intein, and ligating these proteins. A similar method for producing a cyclic or polymerized protein is provided. Modified inteins engineered to cleave at their C-terminus or N-terminus, respectively, and DNA and plasmids encoding these modified inteins are also provided.